UDP-GlcNAc transport across the Golgi membrane: electroneutral exchange for dianionic UMP

Abstract

We have examined the coupling and charge stoichiometry for UDP-GlcNAc transport into Golgi-enriched vesicles from rat liver. In the absence of added energy sources, these Golgi vesicles concentrate UDP-GlcNAc at least 20-fold, presumably by exchange with endogenous nucleotides. Under the conditions used, extravesicular degradation of UDP-GlcNAc has been eliminated, and less than 15% of the internalized radioactivity becomes associated with endogenous macromolecules. Of the remaining intravesicular label, 85% remains unmetabolized UDP-[3H]GlcNAc, and approximately 15% is hydrolyzed to [3H]GlcNAc-1-phosphate. Efflux of accumulated UDP-[3H]GlcNAc is induced by addition of nonradioactive UDP-GlcNAc, UMP, UDP, or UDP-galactose to the external medium. Permeabilization of Golgi vesicles causes a rapid and nearly complete loss of internal UDP-[3H]GlcNAc, indicating that the results reflect transport and not binding. Moreover, transport of UDP-[3H]GlcNAc into these Golgi vesicles was stimulated up to 5-fold by mechanically preloading vesicles with either UDP-GlcNAc or UMP. The response of UMP/UMP exchange and UMP/UDP-GlcNAc exchange to alterations in intravesicular and extravesicular pH suggests that UDP-GlcNAc enters the Golgi apparatus in electroneutral exchange with the dianionic form of UMP.